Figure 4. The frequency of circulating melanoma cells in the blood of xenografted NSG mice correlates with metastatic efficiency.

Blood was collected from untransplanted control or melanoma-transplanted experimental NSG mice by cardiac puncture. Live nucleated blood cells were analysed for HLA⁺ melanoma cells by flow cytometry. Blood from mice bearing an efficiently metastasizing melanoma (A; melanoma 405) and an inefficiently metastasizing melanoma (B; melanoma 528). HLA⁺ cells were not detected in the blood of untransplanted control mice (A, <0.01% background staining) or in the blood of mice bearing inefficiently metastasizing melanomas (B, <0.01% background staining) but were detected in the blood of mice bearing efficiently metastasizing melanomas (A, 0.02–0.51%). Note that melanoma 528 (B) was analysed side-by-side with melanoma 405 (A) such that the negative control in panel (A) applied to both analyses. (C) The frequency of HLA+ melanoma cells in the blood of all mice. The difference between mice bearing efficiently-metastasizing (n=15 mice transplanted with melanomas 405, 481, and 633) and inefficiently metastasizing melanomas (n=7 mice transplanted with melanomas 528 and 651) was statistically significant (p=0.0023 by Anova comparing efficient metastasizers to both other treatments; p=0.0004 by post-hoc pairwise Mann-Whitney t-tests). (D) Several of the mice with inefficiently metastasizing melanomas had large tumor burdens and yet still did not have detectable levels of circulating melanoma cells in their blood (see C). (E) NSG mice were injected subcutaneously with 100 HLA+/GFP+ cells from the blood of NSG mice with subcutaneous melanomas. Palpable subcutaneous tumors developed within 2 months after injection. Mice were analysed by BLI for metastases when the subcutaneous tumor reached 2cm in diameter. Widespread metastasis was observed, just as in the primary melanomas from which the circulating melanoma cells derived.